Hyperglycemia is very common in patients with sepsis even if there is no history of diabetes. Insulin resistance of skeletal muscle glucose uptake (MGU) is a major cause of this hyperglycemia. Control of MGU is distributed between delivery of glucose to muscle, glucose transport into muscle, and glucose phosphorylation within muscle; insulin resistance is due to defects in one or more of them. The impact of obesity on MGU has been studied by a number of groups, but the impact of inflammation on the distribution of control of MGU is unknown. The experiments described in this proposal will examine the extent to which inflammation induced by lipopolysaccharide (LPS) redistributes the control of MGU. In this proposal the roles transport and phosphorylation play in controlling MGU will be assessed by using germline manipulation (partial knockout or over expression) of transport and phosphorylation capacity (e.g. hexokinase) to modulate a single step or multiple steps and measure the impact on MGU. We hypothesize that defects in glucose phosphorylation capacity play a central role in the inflammation induced insulin resistance. Experiments will be performed in chronically catheterized, conscious mice. This approach allows for comprehensive metabolic assessment of MGU in vivo in the absence of stress. The experimental strategy is to perturb proteins or processes involved in control of MGU and measure the effect of the perturbation on glucose influx. Whole body glucose uptake and MGU will be measured using [3-3H] glucose and [14C] 2- deoxyglucose, respectively, in combination with methods for sampling blood and tissues and measuring muscle blood flow. The relationship of MGU to long chain fatty acid (LCFA) uptake will simultaneously be measured using a radiolabeled fatty acid analog. Muscle ATP flux will be assessed using 31NMR spectroscopy. Tissues will be analyzed for glycogen synthesis, insulin signaling, oxidative stress and GLUT4 translocation. Our specific aims are to determine: 1. The impact of LPS on the relative control transport and glucose phosphorylation have in determining MGU 2. If LPS amplifies the impact NEFA and glucose availability have in modulating MGU 3. If modulating oxidative stress (NO availability and NF-:B activation) following LPS will improve MGU by augmenting glucose phosphorylation and mitochondrial ATP flux Our long term goal is to identify the steps controlling MGU that are impacted by inflammation and assess which of those steps are more responsive to changes in oxidative stress. Future therapies can then have a more targeted approach in correcting MGU during an inflammatory stress such as sepsis. PUBLIC HEALTH RELEVANCE: Hyperglycemia is very common in hospitalized patients and clinical trials suggest if the hyperglycemia can be minimized morbidity and mortality are improved. A major cause of the hyperglycemia is insulin resistance of skeletal muscle glucose uptake This proposal will determine where the defect is and address the questions is it due simply to a failure of insulin to activate it signaling pathway or are underlying defects in the mitochondria caused by the inflammation aggravating and limiting the ability of insulin to exert is beneficial effects.